# US 2025/0145976 A1 · Messenger rna encoding cas9 for use in genome-editing systems > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2025/0145976 A1 - Document ID: US20250145976A1 - Status: Published application - Kind: A1 - Application number: 19/008,854 - Filed: 2025-01-03 - Published: 2025-05-08 - Earliest claimed: 2020-05-15 - Assignees: Crispr Therapeutics AG - Applicants: Crispr Therapeutics AG - Inventors: Christian Cobaugh - Web page: https://patentgrep.com/patent/US20250145976A1 ## CPC - C12N 9/226: {Class 2 CAS enzyme complex, e.g. single CAS protein} - C12N 15/111: {General methods applicable to biologically active non-coding nucleic acids} - A61K 9/5123: {Organic compounds, e.g. fats, sugars} - A61K 31/7088: Compounds having three or more nucleosides or nucleotides - A61K 48/005: {characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered} - C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]} - C12N 15/102: {Mutagenizing nucleic acids} - C12N 15/11: DNA or RNA fragments; Modified forms thereof (DNA or RNA not used in recombinant technology, C07H21/00); {Non-coding nucleic acids having a biological activity} - C12N 15/88: using microencapsulation, e.g. using {amphiphile} liposome vesicle - C12N 15/907: {in mammalian cells} - A61K 48/00: Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy - C12N 2310/20: involving clustered regularly interspaced short palindromic repeats [CRISPR] - C12N 2310/335: Modified T or U - C12N 2320/32: Special delivery means, e.g. tissue-specific - C12N 2800/80: Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D) ## Abstract The present disclosure provides optimized mRNAs encoding a site-directed endonuclease for use in a CRISPR/Cas system. Also provided herein are delivery systems for use of the CRISPR/Cas system in methods of in vivo and ex vivo genome editing. ## Claims ### Claim 1 An mRNA comprising: a 5′ untranslated region (UTR); an open reading frame (ORF) comprising a nucleotide sequence that encodes a site-directed endonuclease, wherein the nucleotide sequence is at least 85% identical to a nucleotide sequence of SEQ ID NO: 4; and a 3′ untranslated region (UTR). ### Claim 2 (depends on 1) The mRNA of claim 1, wherein the mRNA comprises at least one chemically modified nucleoside. ### Claim 3 (depends on 2) The mRNA of claim 2, wherein the chemically modified nucleoside is selected from pseudouridine, N1-methylpseudouridine, and 5-methoxyuridine. ### Claim 4 (depends on 2, 3) The mRNA of claim 2 or 3, wherein the chemically modified nucleoside is N1-methylpseudouridine. ### Claim 5 (depends on 1, 2, 3, 4) The mRNA of any one of claims 1-4, wherein at least about 80% of the uridines are chemically modified. ### Claim 6 (depends on 1, 2, 3, 4, 5) The mRNA of any one of claims 1-5, wherein 100% of the uridines are chemically modified. ### Claim 7 (depends on 5, 6) The mRNA of claim 5 or 6, wherein the uridines are modified and/or replaced with N1-methylpseudouridine. ### Claim 8 An mRNA comprising: a 5′ UTR; an ORF comprising a nucleotide sequence that encodes a site-directed endonuclease, wherein the nucleotide sequence is at least 85% identical to the nucleotide sequence of SEQ ID NO: 4; and a 3′ UTR, wherein 100% of the uridines of the mRNA are modified and/or replaced with N1-methylpseudouridine. ### Claim 9 (depends on 1, 2, 3, 4, 5, 6, 7, 8) The mRNA of any one of claims 1-8, wherein the 5′ UTR comprises a nucleotide sequence of SEQ ID NO: 10 or SEQ ID NO: 15. ### Claim 10 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9) The mRNA of any one of claims 1-9, wherein the 3′ UTR comprises a nucleotide sequence of SEQ ID NO: 12. ### Claim 11 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) The mRNA of any one of claims 1-10, wherein the mRNA further comprises a poly-A tail. ### Claim 12 (depends on 11) The mRNA of claim 11, wherein the poly-A tail is about 100 to about 1000, about 10 to about 500, about 10 to about 300, about 10 to about 200, about 50 to about 200, about 50 to about 150, about 100 to about 150, or about 120 to about 150 adenosine nucleotides. ### Claim 13 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) The mRNA of any one of claims 1-12, wherein the mRNA comprises the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 14. ### Claim 14 An mRNA comprising a nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 14, wherein 100% of the uridines of the mRNA are modified and/or replaced with N1-methylpseudouridine. ### Claim 15 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14) The mRNA of any one of claims 1-14, wherein the mRNA comprises a 5′ cap. ### Claim 16 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15) The mRNA of any one of claims 1-15, wherein the 5′ cap is a cap-0, a cap-1, or a cap-2 structure. ### Claim 17 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16) A system for introducing a double-stranded DNA break (DSB) in a target gene in a cell, the system comprising: (a) the mRNA of any one of claims 1-16; and (b) at least one guide RNA (gRNA) directed to the target gene, wherein the mRNA is translated when the mRNA contacts the cell and provides a site-directed endonuclease that combines with the gRNA to induce a DSB at a site in the target gene. ### Claim 18 (depends on 17) The system of claim 17, wherein the mRNA and the gRNA are individually formulated in a lipid nanoparticle (LNP). ### Claim 19 (depends on 17) The system of claim 17, wherein the mRNA and the gRNA are co-formulated in a lipid nanoparticle (LNP). ### Claim 20 (depends on 17, 18, 19) The system of any one of claims 17-19, wherein the LNP comprises one or more lipid moieties selected from: an amino lipid, an ionizable lipid, a neutral lipid, a PEG-lipid, a helper lipid, a cholesterol or derivative thereof. ### Claim 21 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16) A system for correcting a mutation in a target gene in a cell, the system comprising: (a) the mRNA of any one of claims 1-16; (b) at least one guide RNA (gRNA) directed to the target gene; and (c) a donor polynucleotide, wherein the mRNA is translated when the mRNA contacts the cell and provides a site-directed endonuclease that combines with the gRNA to induce a DSB at a site in or near the mutation in the target gene, and wherein a non-homologous end-joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into the DSB at a location proximal to the mutation, thereby correcting the mutation. ### Claim 22 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20) A pharmaceutical composition comprising: the mRNA of any one of claims 1-16 or the system of any one of claims 17-20, and a pharmaceutically acceptable carrier. ### Claim 23 (depends on 22) The pharmaceutical composition of claim 22, further comprising at least one gRNA directed to the target gene. ### Claim 24 (depends on 22, 23) The pharmaceutical composition of claim 22 or 23, further comprising a donor polynucleotide. ### Claim 25 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 23, 24) A method for inducing a DSB in a target gene in a cell, the method comprising: contacting the cell with: (i) the mRNA of any one of claims 1-16 and at least one gRNA directed to the target gene; (ii) the system of any one of claims 17-21; or (iii) the pharmaceutical composition of claim 23 or 24, wherein the mRNA is translated when the mRNA, the system, or the composition contacts the cell and provides a site-directed endonuclease that combines with the gRNA to induce a DSB at a site in the target gene. ### Claim 26 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 23, 24) A method of treating a patient with a disease by inducing a DSB in a target gene in a cell, the method comprising: isolating a cell from the patient, and contacting the cell with: (i) the mRNA of any one of claims 1-16 and at least one gRNA directed to the target gene; (ii) the system of any one of claims 17-21; or (iii) the pharmaceutical composition of claim 23 or 24, wherein the mRNA is translated when the mRNA, system, or composition contacts the cell and provides a site-directed endonuclease that combines with the gRNA to induce a DSB at a site in the target gene. ### Claim 27 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 23, 24) A method of treating a patient with a disease by inducing a DSB in a target gene in a cell, the method comprising: administering to the patient an effective amount of: (i) the mRNA of any one of claims 1-16 and at least one gRNA directed to the target gene; (ii) the system of any one of claims 17-21; or (iii) the pharmaceutical composition of claim 23 or 24, wherein the mRNA is translated when the mRNA, system, or composition contacts the cell and provides a site-directed endonuclease that combines with the gRNA to induce a DSB at a site in the target gene. ### Claim 28 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16) A method for correcting a mutation in a target gene in a cell, the method comprising: contacting the cell with the mRNA of any one of claims 1-16, at least one gRNA directed to the target gene, and a donor polynucleotide, wherein the mRNA is translated when the mRNA contacts the cell and provides a site-directed endonuclease that combines with the gRNA to induce a DSB at a site in or near the mutation in the target gene, and wherein a non-homologous end-joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into the DSB at a location proximal to the mutation, thereby correcting the mutation. ### Claim 29 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16) A method of treating a patient with a disease by correcting a mutation in a target gene in a cell, the method comprising: isolating a cell from the patient; and contacting the cell with the mRNA of any one of claims 1-16, at least one gRNA directed to the target gene, and a donor polynucleotide, wherein the mRNA is translated when the mRNA contacts the cell and provides a site-directed endonuclease that combines with the gRNA to induce a DSB at a site in or near the mutation in the target gene, and wherein a non-homologous end-joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into the DSB at a location proximal to the mutation, thereby correcting the mutation. ### Claim 30 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16) A method of treating a patient with a disease by correcting a mutation in a target gene in a cell, the method comprising: administering to the patient an effective amount of the mRNA of any one of claims 1-16, at least one gRNA directed to the target gene, and a donor polynucleotide, wherein the mRNA is translated when the mRNA contacts the cell and provides a site-directed endonuclease that combines with the gRNA to induce a DSB at a site in or near the mutation in the target gene, and wherein a non-homologous end-joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into the DSB at a location proximal to the mutation, thereby correcting the mutation. ### Claim 31 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24) A kit for inducing a DSB in a target gene in a cell, the kit comprising: a container comprising an mRNA of any one of claims 1-16, the system of any one of claims 17-21, or the pharmaceutical composition of claim 22-24, and a package insert comprising instructions for use.